Autophagy inhibition improves the targeted radionuclide therapy efficacy of 131I-FAP-2286 in pancreatic cancer xenografts

Purposes Radiotherapy can induce tumor cell autophagy, which might impair the antitumoral effect. This study aims to investigate the effect of autophagy inhibition on the targeted radionuclide therapy (TRT) efficacy of 131I-FAP-2286 in pancreatic cancer. Methods Human pancreatic cancer PANC-1 cells were exposed to 131I-FAP-2286 radiotherapy alone or with the autophagy inhibitor 3-MA. The autophagy level and proliferative activity of PANC-1 cells were analyzed. The pancreatic cancer xenograft-bearing nude mice were established by the co-injection of PANC-1 cells and pancreatic cancer-associated fibroblasts (CAFs), and then were randomly divided into four groups and treated with saline (control group), 3-MA, 131I-FAP-2286 and 131I-FAP-2286 + 3-MA, respectively. SPECT/CT imaging was performed to evaluate the bio-distribution of 131I-FAP-2286 in pancreatic cancer-bearing mice. The therapeutic effect of tumor was evaluated by 18F-FDG PET/CT imaging, tumor volume measurements, and the hematoxylin and eosin (H&E) staining, and immunohistochemical staining assay of tumor tissues. Results 131I-FAP-2286 inhibited proliferation and increased the autophagy level of PANC-1 cells in a dose-dependent manner. 3-MA promoted 131I-FAP-2286-induced apoptosis of PANC-1 cells via suppressing autophagy. SPECT/CT imaging of pancreatic cancer xenograft-bearing nude mice showed that 131I-FAP-2286 can target the tumor effectively. According to 18F-FDG PET/CT imaging, the tumor growth curves and immunohistochemical analysis, 131I-FAP-2286 TRT was capable of suppressing the growth of pancreatic tumor accompanying with autophagy induction, but the addition of 3-MA enabled 131I-FAP-2286 to achieve a better therapeutic effect along with the autophagy inhibition. In addition, 3-MA alone did not inhibit tumor growth. Conclusions 131I-FAP-2286 exposure induces the protective autophagy of pancreatic cancer cells, and the application of autophagy inhibitor is capable of enhancing the TRT therapeutic effect. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-024-04958-6.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) remains the tumor with the poorest prognosis in the digestive tract, with a 5-year survival rate less than 10%, and is predicted to become the second leading cause of cancer-related death in western countries by 2030 [1].Although surgical resection currently represents the best opportunity for cure and long-term survival, only up to 20% of patients present at a tumor stage suitable for resection [2].Given that most of PDAC are diagnosed at a late stage with regional metastases and/or distant metastases, finding the proper treatment method is necessary to improve the dismal prognosis [3].
As a special form of radiotherapy, targeted radionuclide therapy (TRT) of tumor is based on the use of radiolabeled peptides or antibodies, which serve as the molecular carriers of radionuclides and bind to the overexpressed receptors or specific antigens in tumor cells [4].Unlike external beam therapy, TRT allows the targeted radiopharmaceuticals delivery to the clinically diagnosed tumor site as well as the metastasized tumor cells, thus benefiting patients with late-stage and metastatic disease [5].Therefore, TRT offers a new therapeutic option for PDAC.Some clinical trials of pancreatic cancer treatment have demonstrated the safety and potential efficacy of TRT, yet the therapeutic effect still need to be improved [6].
Fibroblast activation protein (FAP) is specifically overexpressed in pancreatic cancer-associated fibroblasts (CAFs), which are the major cell component of the tumor microenvironment in pancreatic cancer [7].This selective expression provides a potential therapeutic target for TRT.In fact, radionuclide-labeled FAP inhibitors (FAPIs) have been developed for the targeted imaging or therapy of various types of cancers [8][9][10].However, FAPIs with a short tumor retention time are unfavorable for TRT [11,12].FAP-2286 is a FAP-binding cyclic peptide, and exhibits prolonged tumor retention, which is an important prerequisite for TRT [13][14][15]. 131I with a relative long half-life of 8 days is a clinically used therapeutic radionuclide, allowing for prolonging radiation exposure and matching the tumor retention capabilities of FAP-2286 [16].Based on these properties, 131 I-labeled FAP-2286 may serve as a good TRT agent for pancreatic cancer, and certainly, strategies to further improve the therapeutic effect is warranted.
Generally, radiotherapy can cause cancer cell death through intrinsic, autonomous modalities directly triggered in the irradiated cells (e.g., apoptosis, autophagic cell death, mitotic catastrophe), or extrinsic, non-cell autonomous modalities mediated by the microenvironment or immune system (e.g., senescence, immunogenic cell death).However, the tumor cells also survive via the activation of the survival pathways, such as DNA damage repair, cell cycle arrest and autophagy, which allow cells to endure damage [17].Autophagy of cancer cells is frequently observed during radiotherapy [18,19].Interestingly, pancreatic CAFs also has the ability to suppress the radiotherapy effect via promoting the autophagy of the irradiated pancreatic cancer cells [20,21].Therefore, it is desired to investigate whether 131 I-FAP-2286 TRT affects the autophagy of pancreatic cancer cells, and whether the autophagy inhibitors help improve the therapeutic efficacy of TRT.
In this study,

The synthesis and in vitro stability of 131 I-labeled FAP-2286-Tyr
For labeling FAP-2286-Tyr with 131 I, a simple Iodogen method was used.In brief, 20 μL 131 I-NaI solution (9.5 MBq/μL) and 100 μL PBS buffer containing 20 μg FAP-2286-Tyr were added into the Iodogen tube pre-coated with 50 μg Iodogen orderly.Iodogen tube was placed on vortex mixer at room temperature for 10 min, and then the developed 131 I-FAP-2286 in the supernatant liquid was removed from the reaction tube.
The labeling rate of 131 I-FAP-2286 was tested using radio instant thin-layer chromatography (radio-iTLC) method in which Whatman chromatographic strip paper and 0.9% NaCl (normal saline) was used as the stationary phase and mobile phase, respectively.The in vitro stability test was performed by mixing the labeled product with PBS, 10% FBS or normal saline, respectively, at 4 °C and 37 °C, and incubating for 1, 2, 4, 8, 12 and 24 h before determining the radiochemical purity.

Western blot analysis
The expression of p62, a classical marker of autophagy, in PANC-1 cells exposed to 131 I-FAP-2286, was measured by western blotting.To assess the influence of CAFs on the autophagy of PANC-1 induced by 131 I-FAP-2286, the cell co-culture experiment was performed in a 6-well plate with 3 μm pore size transwell inserts, where PANC-1 cells and CAFs were seeded into the bottom wells and the upper inserts, respectively.After a 24-h culture with 131 I-FAP-2286, p62 expression in PANC-1 was detected.After receiving the corresponding treatments, the cells were lysed using RIPA lysis buffer in the presence of protease and phosphatase inhibitors (Minitab, Roche).The lysates were sonicated on ice and then centrifuged at 12,000 rpm at 4 °C for 10 min.The supernatants were collected for the protein concentration detection, and then were exposed to 12% SDS-polyacrylamide gel electrophoresis.
The separated proteins were transferred to nitrocellulose membranes, followed by being blocked with Odyssey blocking buffer (LI-COR) in PBS containing 1% Tween-20.Primary antibodies of p62 and β-actin were incubated in blocking buffer at 4 °C overnight, and then were detected using HRP-conjugated secondary antibody.The photos of the target protein bands were captured using LI-COR Odyssey protein imaging system and quantified by Image J software.

Immunofluorescence (IF)
PANC-1 cells were cultured on coverslips in 6-well plates, and then were incubated for 24 h after receiving the responding treatments.After being fixed for 5 min at − 20 °C with ice-cold methanol, cells were blocked for 1 h at room temperature with 5% BSA, followed by incubation with primary antibodies (LC3II, p62) overnight at 4 °C.Following that, the cells were rinsed with PBS and incubated for 1 h at room temperature with Alexa Fluor 488-conjugated secondary antibodies.Nuclei were stained with DAPI for 10 min at room temperature.The cells on the slides were observed under a fluorescence microscope (magnification, ×100; BX53, Olympus Corporation).

Transmission electron microscopy (TEM)
PANC-1 cells were collected and centrifuged at 1200 rpm for 5 min.The supernatant was discarded, and then the cells were quickly fixed with electron microscope fixation solution at 4 °C for 2 h.After this, the cell samples were transported to Servicebio Biotechnology Co., Ltd (Wuhan, China), and the sample preparation process for the conventional TEM was carried out.Finally, 60 nm ultrathin sections were obtained and observed using Hitachi-7500 transmission electron microscope (Hitachi, Japan).

Calcein-AM/PI assay
After treatment, PANC-1 cells were added with Calcein-AM/PI staining solution and incubated at 37 °C for 20 min following the manufacturer instructions.Under the fluorescence microscope, the living cells and dead cells were identified as green fluorescence and red fluorescence, respectively.

Apoptosis detection
Cell apoptosis was determined using an Annexin-V-FITC/propidium iodide (PI) apoptosis kit.PANC-1 cells upon different treatments were cultured in 6-well culture plates, Cells were collected and washed twice with icecold PBS before double staining with Annexin V-FITC/PI after 24 h.Apoptosis detection was performed in a flow cytometer and the results were analyzed using the Cell-Quest Pro (IVD) software (BD, Bioscience).

I-FAP-2286 TRT of pancreatic cancer xenograft-bearing nude mice
All animal experiments were approved by the Ethics Committee of Naval Medical University, and were conducted with the guidance of ethical principles governing animal welfare, rearing, and experimentation.Female nude mice (age, 4 weeks) were purchased from Vital River Laboratory Animal Technology Co., Ltd, and were raised under specific pathogen-free (SPF) conditions.For the establishment of subcutaneous xenograft pancreatic cancer model in nude mice, 100 μL of cell suspension of PANC-1 (5 × 10 6 ) with CAFs (5 × 10 6 ) or PANC-1 (5 × 10 6 ) alone were injected into the right upper limb, respectively.The size of tumor was measured every other day.When the tumor volume reached about 60 mm 3

SPECT/CT imaging of pancreatic cancer xenograft-bearing nude mice
In this experiment, free-[ 131 I] or 131 I-FAP-2286 (9.25 MBq/200 μL) was injected into pancreatic cancer xenograft-bearing nude mice (PANC-1 alone, or PANC-1 + CAFs co-injection) via tail vein, and then SPECT/CT imaging was performed at different time points post injection.For tomography with high-energy high-resolution collimator, matrix: 64 × 64; zoom: 2; energy peak: 35 keV; window width: 20%; frame size: 60 s/frames; total acquisition of 32 frames.For CT, tube voltage: 130 kV; tube current: 35 mA; pitch: 1.0; reconstructed layer thickness: 1 mm.3D regions of interest (ROIs) were drawn over the whole-body, brain, thyroid, lungs, heart, liver, spleen, intestine, muscle and tumor on decay-corrected whole-body images to obtain the total counts and volume.The biodistribution were expressed as %ID/mL, which represents the percentage injected dose per milliliter of tissue.

F-FDG PET/CT imaging
For the pancreatic cancer xenograft-bearing nude mice, 18 F-FDG PET/CT imaging was performed before and after treatment (n = 3 per group).Mice were fasted for 8 h and then were administered with 7.4 MBq of 18 F-FDG via the tail vein.About 1 h later, PET/CT imaging was carried out.Maximum standardized uptake value (SUV max ) was calculated by the TrueD system automatically by drawing ROI.

Statistical analysis
Data are presented as the mean ± SD. Analysis of variance (ANOVA) and Student's t-test were used for the comparisons among groups.P value less than 0.05 was considered statistically significant.All experiments were performed in triplicate.

I-FAP-2286 exposure induces autophagy in PANC-1 cells
131 I-FAP-2286 exposure induced the proliferation inhibition of PANC-1 cells in a radioactive dose-dependent manner (Fig. 2A).P62 is a well-established autophagy marker, whose expression is inversely correlation with the autophagy level.As shown in Fig. 2B, C, the expression of p62 in PANC-1 cells was reduced gradually along with the radioactive concentration increasement of 131 I-FAP-2286, indicating that the cell autophagy level was elevated.Additionally, under the condition of indirect coculture with CAFs, p62 expression in PANC-1 cells was further decreased after 131 I-FAP-2286 exposure (Additional file 1: Fig. S1), meaning that CAFs enhanced the radiation-induced autophagy of PANC-1 cells.Notably, 9.25 MBq/mL of 131 I-FAP-2286 exhibited mild cytotoxicity, but produced obvious autophagic effect on PANC-1 cells.Thus, 9.25 MBq/mL as a representative concentration was chosen for the subsequent experiments.

Autophagy inhibitor reverses the elevated autophagy level in PANC-1 cells induced by 131 I-FAP-2286
TEM images revealed that more cytoplasmic autophagosomes appeared in 131 I-FAP-2286-treated PANC-1 cells compared to controls, but this phenomenon was reversed by the autophagy inhibitor 3-MA (Fig. 3A).Western blot experiments shown that, in 131 I-FAP-2286 treatment group, the expression of p62 was lowered, but was restored by the application of 3-MA (Fig. 3B, C).Consistently, the average p62 fluorescence intensity per cell  3D and E).LC3II is another autophagy marker, whose expression is positively correlated with autophagy level.As shown in Fig. 3D and F, the mean fluorescence intensity per cell of LC3II increased significantly in 131 I-FAP-2286 group, but the addition of 3-MA can efficiently block this effect.Together, the above results indicated that 3-MA inhibited the elevated autophagy of PANC-1 cells induced by 131 I-FAP-2286.

Autophagy inhibitor promotes the apoptosis of PANC-1 cells induced by 131 I-FAP-2286
As shown in Fig. 4A, 3-MA treatment alone did not influence the viability of PANC-1 cells.However, 131 I-FAP-2286 significantly inhibited the proliferation of PANC-1 cells, and this effect was further amplified by 3-MA.Calcein-AM/PI staining results showed that 131 I-FAP-2286 combined with 3-MA treatment induced more PANC-1 cells death compared to monotherapy group (Fig. 4B).This result was further confirmed by the apoptosis detection, which showed that 3-MA promoted 131 I-FAP-2286-induced PANC-1 cells apoptosis (Fig. 4C, D).Notably, 3-MA treatment alone did not cause obvious apoptosis.Taken together, these results demonstrated that autophagy inhibitor 3-MA enhanced 131 I-FAP-2286-triggered apoptosis in PANC-1 cells., S3).As shown in Fig. 5B, 131 I-FAP-2286 was mainly concentrated in the kidney and liver at 1 h after intravenous injection, suggesting that 131 I-FAP-2286 was primarily excreted through the urinary system.The radioactivity in the thyroid and stomach was low, meaning that 131 I-FAP-2286 is relatively stable to avoid in vivo deiodination.In addition, the tumor uptake of 131 I-FAP-2286 in the blocking group was significantly lower than that in the non-blocking group ((0.0067 ± 0.0001%ID/ mL) vs (0.0233 ± 0.0026%ID/mL), P = 0.032) (Fig. 5C).According to the target-to-normal tissue ratio (TNR), the maximum value occurred at 18 h post-injection of 131 I-FAP-2286 (Fig. 5D).Notably, compared to the tumor with PANC-1 injection alone, the tumor with the coinjection of PANC-1 and CAFs had a stronger positive expression of FAP and α-SMA (Additional file 1: Fig. S4), indicating that it was CAFs that provided the target for 131 I-FAP-2286.

Autophagy inhibition enhances the TRT efficacy of 131 I-FAP-2286 in pancreatic cancer
The 18 F-FDG uptake reflects the activity of tumor.As shown in Fig. 6A, B, compared to control group, 131 I-FAP-2286 TRT markedly reduced the uptake of 18 F-FDG in tumor (SUV max : 2.29 ± 0.16 vs 1.68 ± 0.08, P < 0.01), but the lowest SUV max of tumor was observed in 131 I-FAP-2286 + 3-MA group.Besides, the SUV max in 3-MA group was equivalent to the control group.These results indicated that 131 I-FAP-2286 TRT was capable of curbing pancreatic tumor growth, and the addition of autophagy inhibitor 3-MA could enhance the therapeutic effect.
Being consistent with the results of 18 F-FDG PET/CT, 131 I-FAP-2286 TRT significantly inhibited the tumor growth, and 3-MA alone did not affect the tumor growth.Notably, the combination treatment of 131 I-FAP-2286 and 3-MA displayed the strongest anti-tumor effect (Fig. 7A,  B).In addition, H&E staining assay showed that 131 I-FAP-2286 TRT increased the area of tumor necrosis and apoptosis, which was more serious in 131 I-FAP-2286 + 3-MA group, but while 3-MA alone did not cause this effect (Fig. 7C).Ki-67 is frequently used to assess the proliferation of tumor cells.As shown in Fig. 7D and F, after 131 I-FAP-2286 TRT, the expression of Ki-67 was obviously down-regulated, meaning that the proliferation of tumor cells was suppressed.It should be noted that, compared to 131 I-FAP-2286 TRT alone, the combination treatment group possessed lower ki-67 expression, signifying better therapeutic effect.
The autophagy level of tumors with different treatment also was detected. 131I-FAP-2286 TRT decreased p62 expression but increased LC3II expression of tumor, confirming that 131 I-FAP-2286 exposure also induced autophagy in vivo.In 131 I-FAP-2286 + 3-MA group, the TRT-mediated autophagy of pancreatic tumors was restored (Fig. 7E, G, H).The above results suggested that the enhanced anti-tumor effect of the combination treatment of 131 I-FAP-2286 and 3-MA could be attributed to the inhibition of autophagy.efficiently target the tumor and exert TRT effect along with the autophagy induction.The combination of 3-MA and 131 I-FAP-2286 achieved a better therapeutic effect, which was attributed mainly to the destruction of the protective autophagy of tumor (Fig. 9).As an effective radiotherapy approach, TRT has demonstrated considerable promise in various types of cancer, including pancreatic cancer [22,23].For the TRT of pancreatic cancer, the specificity of pancreatic cancer biomarkers, such as FAP, is conducive to the improvement of the radiopharmaceutical uptake ratio of tumorto-normal tissue [24][25][26][27].FAP-2286 has an advantage of accumulating in the FAP-positive tumor tissues for a relative long time [28,29]. 131I-FAP-2286 can specifically target CAFs in the tumor stroma, and further irradiates tumor cells via non-specific cross-irradiation [30,31].According to the immunohistochemistry assay, the expression of α-SMA, a well-known marker of CAFs, was consistent with FAP in the pancreatic cancer xenograft model with the co-injection of PANC-1 and CAFs, confirming the presence of abundant FAP-positive CAFs.Our results confirmed that 131 I-FAP-2286 was rapidly cleared from non-target organs except for kidney and remained in pancreatic cancer xenografts more than 96 h, which was the important premise for the TRT.From the view point of the safety of TRT, using lower radioactive dose for better therapeutic effect is the everlasting pursuit.Therefore, validating the feasibility of 131 I-FAP-2286 TRT for pancreatic cancer just is the first step.
Numerous studies have shown that radiation exposure can activate the protective autophagy of tumor cells, which may produce negative effect on the efficacy of radiotherapy [20,32].Radiotherapy induced ROS-dependent autophagy in macrophages through unfolded protein response [33]. 131I-FAP-2286 TRT may also face a similar situation.Our results demonstrated that 131 I-FAP-2286 reduced p62 expression and promoted the formation of autophagosomes, meaning the occurrence of the protective autophagy.Within a certain dose range, 131 I-FAP-2286 exposure induced autophagy in a dose-dependent manner.Autophagy inhibitor 3-MA blocks the early process of radiationinduced autophagy by impairing the formation of the PI3K complex [34,35].Furthermore, it has been reported that 3-MA can induce caspase-dependent cell death without the aid of autophagy inhibition [36].When the autophagy was inhibited by 3-MA, 131 I-FAP-2286 displayed a stronger anti-tumor ability, suggesting that blocking the autophagy activation of pancreatic cancer cells decrease their ability to resist radiation damage.It has been reported that autophagy inhibition has the potential to enhance the radio-sensitivity of radiation-resistant pancreatic cancer [37,38].According to our present result, suppression of autophagy has a promotional effect on 131 I-FAP-2286 TRT, and the combination of autophagy inhibitor and TRT is expected to make for better outcomes via inducing multiple types of tumor cell death simultaneously, offering a unique approach to enhance the efficacy of radiotherapy [39].
Studies have shown that tumor cells can enter a senescent or dormant state, and further become resistant to radiotherapy [40,41].Autophagy inhibition has the ability of destroying the senescence and dormancy of tumor cells, which also may be a potential mechanism of improving the therapeutic efficacy.The future research will focus on elucidating the underlying molecular mechanisms of TRT-induced autophagy, as well as the synergistic effect of TRT in combination with autophagy inhibition.Our present findings provide a reference for the preclinical study of 131 I-FAP-2286 TRT combined with autophagy inhibitors in the FAP-expressed tumors.
In conclusion, our study suggests that autophagy inhibition can significantly improve the TRT efficacy of 131 I-FAP-2286 in pancreatic cancer, providing a novel strategy for pancreatic cancer radiation therapy.

Fig. 8 Fig. 9
Fig. 8 The in vivo toxicity study of 131 I-FAP-2286 + 3-MA.A Body weight change of mice after different treatments.B ALT, AST, BUN and CREA level in the serum of mice at 2 weeks after injection of saline (control) and 131 I-FAP-2286 + 3-MA.C H&E-stained images of major organs including heart, liver, spleen, lung and kidney collected from the control mice and 131 I-FAP-2286 + 3-MA injected mice, respectively